RFLP mapping of rye chromosome 7R reveals a highly translocated chromosome relative to wheat PDF

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RFLP mapping of rye chromosome 7R reveals a highly translocated chromosome relative to wheat 0. A . ROGNLI,'K. M. DEVOS,C . N. CHINOY,R. L. HARCOURT,M. D. ATKINSON,AND M. D. GALE^ Cambridge Laboratory, Colney Lane, Nor wich NR4 7UJ, U. K. Corresponding Editor: J. P. Gustafson Received April 28, ...

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RFLP mapping of rye chromosome 7R reveals a highly translocated chromosome relative to wheat 0. A . ROGNLI,'K. M. DEVOS,C . N. CHINOY,R. L. HARCOURT,M. D. ATKINSON,AND M. D.

GALE^

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Cambridge Laboratory, Colney Lane, Nor wich NR4 7UJ, U. K. Corresponding Editor: J. P. Gustafson Received April 28, 1992 Accepted July 29, 1992 R. L., ATKINSON, M. D., and GALE,M. D. 1992. RFLP ROGNLI,0 . A., DEVOS,K. M., CHINOY,C. N., HARCOURT, mapping of rye chromosome 7R reveals a highly translocated chromosome relative to wheat. Genome, 35: 1026-1031. A RFLP map of rye, Secale cereale, chromosome 7R was constructed using 22 DNA probes including genes coding for leaf acyl carrier protein 111, chloroplast fructose-1,6-bisphosphatase,a-amylase-2, EmBP-1, and a peroxidase. All probes had previously been located in wheat, Triticum aestivum, and a comparison of the wheat and rye maps reveals that chromosome 7R contains four segments that are homologous with segments of chromosomes of four homoeologous groups of wheat, 5L, 4L, 7 s and 7L including the centromere, and 2s. Within these segments the rye and wheat maps are colinear and orientated in the same way relative to the centromere. The implications of rearrangements such as detected in 7R for triticale breeding and interspecific chromosome recombination and manipulation are discussed. Key words: rye, Secale cereale, RFLP, genetic map, chromosome 7R, colinearity. R. L., ATKINSON, M. D., et GALE,M. D. 1992. RFLP ROGNLI,0 . A., DEVOS,K. M., CHINOY,C. N., HARCOURT, mapping of rye chromosome 7R reveals a highly translocated chromosome relative to wheat. Genome, 35 : 1026-1031. Une carte de PLFR du chromosome 7R du seigle, Secale cereale, a CtC dressCe a l'aide de 22 sondes d'ADN, incluant les genes codant la protCine I11 de la feuille qui est porteuse d'un acyle (((acyl carrier protein))), ainsi que la fructose1,6-biphosphatase des chloroplastes, l'a-amylase-2, 1'EmBP-1 et une peroxydase. Toutes les sondes avaient CtC ant& rieurement 1ocalisCes chez le blC, Triticum aestivum, et une comparaison entre les cartes du blC et du seigle a rCvClC que le chromosome 7R contient quatre segments qui sont homologues de segments de chromosomes de quatre groupes homCologues du blC : les 5L, 4L, 7 s et 7L incluant le centromere, et le 2s. A I'intCrieur de ces segments, les cartes du blC et du seigle sont co-linCaires et orientkes de la mCme f a ~ o npar rapport au centromkre. Les implications de rkarrangements tels que ceux dCcelCs chez le 7R pour 1'amClioration du triticale, de mCme que pour la recombinaison et la manipulation de chromosomes interspkcifiques sont discutCes. Mots clks : Secale cereale, PLFR, carte gCnCtique, chromosome 7R, co-linCaritC. [Traduit par la rkdaction]

Introduction Translocation of the rye genome, relative to wheat, was inferred from the ability of individual rye chromosomes to substitute for several wheat chromosomes long before we came to appreciate the extent of homoeology and genetic map colinearity within wheat, Triticum aestivum (2n = 6x = 42), and between wheat and other Triticeae species such as rye, Secale cereale (2n = 2x = 14). The involvement of chromosome 7R in the rearrangements was first demonstrated by Koller and Zeller (1976) who showed that chromosome 4R could partially compensate for the loss of wheat chromosomes in both homoeologous groups 4 and 7. Later, elegant pairing studies in wheat-rye hybrids by Naranjo et al. (1987) and Naranjo and Fernandez-Rueda (1991), and the relative chromosomal locations of molecular and biochemical marker loci in wheat and rye (Liu et al. 1992) provided evidence that 7R carries homoeoloci on the long arm of homoeologous group 5 chromosomes in wheat. Recently, during routine RFLP screening, we identified D N A probes that detected loci on the short arm of wheat homoeologous group 2 chromosomes, but which hybridized to 7R in rye (Fig. 1). This was the motivation for the construction of the detailed 7R map described below. -

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'permanent address: Department of Biotechnological Sciences, Agricultural University of Norway, P.O. Box 5040, 1432 As, Norway. 2 ~ u t h o to r whom all correspondence should be addressed. Printed in Canada / lrnprime au Canada

The significance of this study lies in the fact that cereal cytogeneticists, who have developed various, sophisticated means of inducing recombination between chromosomes of cultivated bread wheat and related species, employ strategies for the transfer of useful genes that assume complete colinearity. Often recombinant plants that are inferior to the wheat recipient genotype are produced, and this is assumed to be an effect of the presence of inferior alleles linked to the target genes. However, if the genomes of related species are characterized by translocations as found here for 7R, then the deleterious nature of interspecific recombinants may often be due to the production of unbalanced deletion-duplication genotypes. If this is indeed the case, future transfers will require the use of detailed maps both to target the correct wheat chromosome and to return recombinants to a euploid state. Materials and methods Genetic stocks Chromosome and arm locations for RFLP loci in wheat were determined using the available cv. Chinese Spring (CS) nullisomictetrasomic (NT) (Sears 1954) and ditelosomic (DT) (Sears and Sears 1978) stocks. Chromosomal locations in rye were determined using the CS/Secale cereale cv. Imperial chromosome addition lines (Driscoll and Sears 1971). Chromosome locations for some known function loci had been previously determined: XFbp, XPepc (Chao et al. 1989a); XAcl3 (Devos et al. 1991b); Xa-Amy-2 (Chao et al. 19896); XEmbp (Devos et al. 1991a); XFed (Chinoy et al. 1991); and XPer (Devos et al. 1992b).

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FIG. 1. Hybridization of PSR928 to (A) HindIII-restricted genomic DNA from the homoeologous group 2 nullisomic-tetrasomic lines of CS and (B) HindIII-restricted genomic DNA from the CS/S.cereale cv. Imperial addition lines. Mapping was carried out in a population of 128 F, plants, or bulked F,-progenies, from a cross between the inbred rye lines DS2 and R x L10 (MasojC and Gale 1991).

DNA clones Random wheat cDNAs (PSR56-PSR163, Chao et al. 1989b), random wheat PstI and EagI gDNAs (PSR311, Harcourt 1992; PSR547-PSR946, Devos et al. 1992a) and a wheat leaf HpaII clone (PSRlO51, Cheung et al. 1992) were investigated in addition to seven known function clones, listed with their source in Table 1.

RFLP and linkage analysis RFLP analysis was carried out as described by Devos et al. (1992a). Linkage analysis, using MAPMAKER version 2.0 being supplied by E.S. Lander, Whitehead Institute of Biomedical Research, Cambridge, Mass., U.S.A., was carried out as described in Devos et al; (1992a).

Results Many of the 28 clones listed in Table 1 had previously been characterized by hybridization against NT, DT, and (or) the CS/Imperial chromosome addition lines (Chao et al. 1989b; Harcourt 1992; Devos et al. 1992b; Liu et al. 1992). These analyses were completed to provide the data shown in Table 1. Twenty-two clones were hybridized to filters carrying DNA of the 128 progenies of the mapping cross, digested with an appropriate restriction enzyme. The remaining clones detected 7R loci that were either monomorphic (Xpsr6.5, Xpsrl09, Xpsr914, and XFed) or were expected to contribute little new information to the overall map (Xpsr169 and XPepc). The approximate positions of Xpsr169 and XPepc could be inferred from colinearity with wheat chromosomes 7B and 7D (Chao et al. 1989b) to lie proximal to Xa-Amy-2 on 7RL.

Genetic map of 7R Figure 2 shows the best map, based on maximum likelihood differences. Thirteen of the 22 loci were unambiguously (LOD > 2.5, P I0.003) ordered and located by two- and three-point linkage analysis, while the remaining 9, identified by an asterisk in Fig. 2, were placed using the multipoint

algorithm. However, other maps, particularly in the densely mapped centromeric region, can be constructed with nearly equal probabilities. All the loci showed Mendelian segregation ratios, except Xpsr690, which gave a small excess of heterozygotes ( P I0.05). Discussion The subsequent discussion is based on two important assumptions. First, it is assumed that the translocations relative to wheat described here characterize Secale cereale and do not simply reflect intervarietal translocations for which the mapping-cross happens to be homozygous. This assumption is founded on the evidence that the translocations are present in several independent rye genotypes. The segments with homoeology to wheat 4L and 7 correspond with the 4R/7R translocation reported to be present in cvs. Dakold and King I1 (Koller and Zeller 1976). Each of the segments with homoeology to wheat 5L, 4L, and 2 s are present in the 7R chromosomes of all three genotypes investigated here, Imperial, DS2, and R x L10. The second assumption is that the primeval or original Triticeae genome is that exemplified by the D genome of wheat. This is, of course, difficult to justify, since that genome could just as easily be a product of translocations itself. However, since in our case the motivation for examining the rye genome in detail is to more easily transfer useful genes from rye into wheat, the assumption is conceptually most convenient. The 7R map is, in many ways, similar to other rye and wheat chromosomal maps constructed in this laboratory, being about 135 cM in length with clustering of points in the region of the centromere (Chao et al. 1989b; Devos et al. 1992a, 1992b; Wang et al. 1992). In fact 15 (54%) of the 28 loci map or can be placed within a 20 cM centromeric linkage block. The key feature is, however, that 7R is highly translocated relative to wheat. Colinearity with wheat The available evidence shows that each of the four rye segments is completely colinear with wheat. The order and

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orientation, relative to the centromere, of Xa-Amy-2 and Xpsr129 are consistent with the wheat homoeologous group 7L map (Chao et al. 1989b; Harcourt 1992), Xpsr928, Xpsrl.50, and Xpsr649 are consistent with the distal segment of the wheat 2 s map (Devos et al. 1992b), XFbp and Xpsr.59 are consistent with the map of wheat group 4L chromosomes (C.N. Chinoy, unpublished), and Xpsrll.5 and Xpsr.567 are consistent with the distal region of the wheat group 5L map (D.X. Xie, unpublished). None of the translocated 7R segments are homoeologous with entire arms of wheat chromosomes. The 5L segment is homoeologous with only the distal portion of wheat 5L. The 4L segment is homoeologous with the proximal region of wheat 4L. The 7L segment lacks a small distal segment of the original 7RL in that the two distal markers, Ep-1 and Xpsrl21, located by Chao et al. (1989b) towards the ends of 7BL and 7DL, are absent from the present 7RL. Endopeptidase, Ep-I, has been located on 6R by Benito et al. (1991), and analysis of the rye addition lines has also shown Xpsrl2l to be present on 6R. The 2 s segment comprises only the distal portion of wheat 2 s (Devos et al. 1992b). Two loci, XEmbp in the 7L segment, and Xpsr946 in the 5L segment, have no known homoeoloci in wheat. However, the two clones, pGCl9 and PSR946, detect a number of nonhomoeologous loci in wheat (see Table I), and thus the occurrence of unpredicted locations in rye is not surprising. Translocation within the rye genome Relative to the wheat homoeologous group 7 chromosomes, chromosome 7R has evolved as the product of multiple translocations . These rearrangements include the 4R/7R translocation first described by Koller and Zeller (1976), although these workers assumed that the breakpoint was in the centromere. The presence on 7R of XAcl3 and Xpsr6.5, located in wheat on 7S, indicate that a small segment, possibly less than 1 cM, of the original short arm remains. The map also confirms the findings of Naranjo et al. (1987), Naranjo and Fernandez-Rueda (1991), and Liu et al. (1992). They provided evidence, from wheat-rye induced chromosome pairing and the relative chromosomal locations of genetic markers, for the presence of a segment on 7RS homoeologous with part of the long arms of wheat group 5 chromosomes. The occurrence of a segment on 7RL with homoeology to wheat group 2 chromosomes is also consistent with the findings by Naranjo and Fernandez-Rueda (1991) that 7RL pairs with the short arms of wheat homoeologous group 2 chromosomes with a frequency of 0.8%. In wheat, Xpsr6.5 and Xpsr.152 map together near the centromere on short arms of the group 7 chromosomes (Chao et al. 1989b; Harcourt 1992). The chromosomal location of Xpsr1.52 is known to be 4R (Liu et al. 1992), so that the translocation breakpoint in rye relative to wheat can be placed between XAcl3 and Xpsr6.5, which remain on 7RS, and Xpsr1.52, which is now located on 4R, having been translocated from 7s.

Xpsr566* Xpsm9 FIG. 2. RFLP map of rye chromosome 7R and relationships with the hexaploid wheat (W) genome. NOTE:The gene orders of adjacent loci marked with an asterisk are preferred over other possible orders with LOD differences 2. d ~ o p ynumber = 2. eFrom Raines et al. (1988). f ~ r o mHansen (1987). gFrom Lazarus et al. (1985). h~rom Guiltinan et al. (1990). 'Rye loci in parentheses have no known wheat homoeoloci; -, no known homoeolocus. Some wheat chromosomal locations for homoeologous sets reflect evolutionary rearrangements within wheat itself: 2A,-,2D, a 2B deletion (Devos et al. 19926); 4AS, 4BL, 4DL); 4AL, 5BL, 5DL, and 7BS, 5BL, 5DL, product of the 4A/5A/7B translocations (Liu et al. 1992). 'From D. Bringloe, unpublished. k ~ r o mRebmann et al. (1991). 'From P. Westhoff, unpublished.

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The wheat-rye chromosome pairing data presented by Naranjo and Fernandez-Rueda (1991) have accurately predicted the composition of 7R; however, the extent of the pairing observed is not consistent with the genetic lengths of the four linkage blocks within rye: 7RS-(5BL, 5DL, 7BS), 3.2%; 7RS-7WS, 0%; 7RL-7WL, 0.1%; and 7RL-2WS, 0.8%. Clearly, homoeology in the distal regions of the chromosome is more conducive to pairing than interstitial homoeology . The present study does not, however, provide evidence for an inversion in the original 4R chromosome prior to its translocation to 7RS, as suggested by Naranjo and Fernandez-Rueda (1991). The order and orientation of markers in the segment of 7RS with homology to wheat group 4L is consistent with that found on 4BL and 4DL. This segment also carries only those homoeoloci remaining after the proposed 4AL-5AL translocation, indicating that the 4AL-5AL and 4RL-5RL interchanges may derive from the same event. Moreover, the segment of 7RS homoeologous to wheat group 5L is precisely that transferred from 5AL to 4AL in wheat, thereby adding more evidence for the coderivation of the A and R genomes. The subsequent transposition of the original 4RL and 5RL, and 2RS segments to 7R appear to have occurred in rye itself.

Implications of translocated chromosomes in rye The implications of .the rearrangements in 7R relative to wheat homologous group 7 chromosomes are far reaching, particularly since this finding implies that several other rye chromosomes have similarly diverged from the standard wheat homoeology by translocation. Furthermore, different, but similarly complex, translocations may well characterize the genomes of other relatives of wheat that are potential donors of useful genes. It is clear that to transfer a gene from 7R to wheat, one must first ascertain the segment of 7R in which the gene lies. Then the transfer, induced by relaxing homoeologous pairing control by any of the various strategies available, must be made to a wheat chromosome that corresponds with the critical segment and is not further translocated itself. Any other transfer will give an unbalanced genotype that will be extremely difficult to retrieve by further recombination. For example a gene on the 5L segment could be transferred to wheat 4BL by induced recombination in the 4RL segment. The resulting transfer would, when homozygous, be deficient for the distal part of 4L and include an additional segment with homoeology to the distal part of 5L. A transfer to 5BL, however, would be balanced and could, if necessary, be further refined by subsequent homoeologous recombination and selection with linked markers. It is possible that much of our relative lack of success in the area of introgression of useful genes from related species to wheat is due to our lack of comprehension of the scale of the rearrangements between alien genomes and wheat. Indeed, many such experiments result in genotypes with phenotypes inferior to that of the wheat genotype for traits other than the object of the transfer' Genetic maps such as greatly aid future transfers, the One presented here providing the is extended the rest of the rye genome and, possibly, to other relatives of wheat. Rearrangements in the rye genome have similar im~lications for triticale breeding where attempts are being made to substitute D genome chromosomes for an equivalent rye

chromosome to correct deficiencies in, or augment, the phenotype of the primary amphiploid. For example, a 7R (7D) substitution would result in serious duplicationdeficiency from which it is unlikely that a competitive variety could be bred.

Conclusions The complex relationship between the rye and wheat genomes exemplified here by RFLP map analysis of 7R using wheat DNA probes may have profound implications for future strategies designed to transfer genes from rye to wheat. It is imperative that the analyses be extended to include the entire rye genome and other potential donors of exotic genes to wheat to determine the extent of the problem. At the same time, however, the mapped markers will provide a tool to screen ideal intragenomic recombinants from potentially deleterious transfers and to minimize the length of critical segments to include little alien material other than the target gene. Acknowledgement The senior author thanks the Norwegian Agricultural Research Council for financial support during the course of this work. Benito, C., Gallego, F.J., Zaragoza, C., Frade, J.M., and Figueiras, A.M. 1991. Biochemical evidence of a translocation between 6RL/7RL chromosome arms in rye (Secale cereale L.). A genetic map of 6R chromosome. Theor. Appl. Genet. 82: 27-32. Chao, S., Raines, C.A., Longstaff, ...